Pluripotent stem cells are self-replicating stem cells that have an ability to differentiate into differentiated cell types, each of which belongs to at least one of ectoderm, mesoderm, and endoderm, and the cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), embryonal carcinoma cells (EC cells), multipotent adult progenitor cells (MAP cells), and adult pluripotent stem cells (APS cells). Among them, ES cells are illustrated as shown below to describe the invention of the present application.
ES cells are a cell population that is derived from pluripotent stem cell clones comprised in an early embryo, and have an ability to differentiate into diverse cell types including germ cells. ES cells can be proliferated while retaining their pluripotency in a defined culture condition. When ES cells thus cultured in such a culture condition are injected into a blastocyst or a morula, chimeric animals having two different genomes are produced. Chimeric animals that have germ cells derived from the ES cells genetically manipulated may be bred with each other to create animal individuals with a manipulated gene. Thus, ES cells are widely utilized to produce transgenic animals including knockout mice wherein the function of a certain gene is altered. On the other hand, the technical approach to induce the differentiation of ES cells in a dish to provide a certain differentiated cells has been developed. Since the approach is applied to human ES cells to provide differentiated cells necessary in cell transplantation therapy, it is expected that ES cells would be used in the medical field in future.
As described above, the approach to proliferate or establish ES cells while retaining their pluripotency (differentiation potency) without differentiating has been developed, in which the culture medium used for the culture of ES cells usually comprises a serum, including specifically a fetal calf serum (FCS), a horse serum, or goat serum. Serums suppress the differentiation of ES cells, and supply various liquid factors to promote the proliferation or establishment of the ES cells. However, the identities of the liquid factors have not been known yet, and the serum varies in quality depending on production lot. Accordingly, it would be required to select an appropriate serum lot by a preparative screening, which demands a large amount of labor.
As another culture approach, ES cells are seeded and cultured in a medium that comprises, as feeder cells, primary cultures of embryonic fibroblasts inactivated to inhibit the proliferation or STO cells. In this approach, the feeler cells are understood to form a matrix for ES cell attachment, as well as to suppress the ES cell differentiation, and to release various liquid factors that promote the ES cell proliferation. Since leukemia inhibitory factor (LIF), which has been known as one of such liquid factors (U.S. Pat. No. 5,187,077) has a potency to suppress the differentiation of ES cells derived from various animals, a combination with a serum, a feeder cell, and LIF are frequently used. Further, it has been suggested that a large amount of the recombinant LIF protein could be added to a serum-containing medium to culture ES cells in a feeder cell-independent manner on a gelatin-coated plate (U.S. Pat. No. 5,166,065).
Use of a serum or a feeder cell as shown above causes major problems for the application of differentiated cells derived from human ES cells to cell transplantation therapy. Specifically, human ES cells have been usually cultured in a medium containing a fetal calf serum using primary cultures of mouse embryonic fibroblasts as feeder cells, and such heterologous biological components as used therein may be necessarily the source of unknown pathogen contamination. Further, transplantation of human cells co-cultured with heterologous cells should be regarded as xenotransplantation, and recipients undergoing such transplantation are forced to live in restricted circumstances according to the draft guideline of the U.S. Food and Drug Administration.
Under the circumstances, it is apparently desirable that pluripotent stem cells such as ES cells to be used in analysis in cell biology and in medical applications should be isolated and cultured using a medium free of pathogen from a heterologous animal and of heterologous cells, specifically a medium which is free of a feeder cell and a serum, which can be artificially prepared, and which comprises the known ingredients.
In response to the demand, many studies have been conducted and proposed. Representative study includes a culture technique wherein a large amount of the recombinant LIF protein is added to a serum-containing medium to culture ES cells in a feeder cell-independent manner on a gelatin-coated plate (U.S. Pat. No. 5,166,065). Further, a culture medium for embryonic stem cells comprising a defined replacement in stead of a serum has been proposed (Japanese Patent Publication (kokai) No. 2001-508302), which allows to culture the cells in the presence of feeder cells in a medium free of serum. However, even if the medium comprising known ingredients which comprises a defined replacement in stead of a serum, is supplemented with a large amount of the recombinant LIF protein, it has been impossible to stably culture ES cells in a feeder cell-independent manner on a gelatin-coated plate. In other words, when cultured using a serum replacement in stead of a serum according to the conventional manner, the feeder cell-independent ES cells have not been capable of being proliferated or established on a gelatin-coated plate under a condition that the cells are seeded at a lower density.
There is a demand to proliferate ES cells even under a condition that the cells are seeded at a lower density in the establishment and the genetic engineering of ES cells, but the demand cannot be satisfied by the approaches as shown above.